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Divisions of Clinical Pharmacology (P.S., L.J.M., S.K., N.J.B.), Pediatric Endocrinology (P.S.), and Cardiovascular Medicine (D.E.V.), Vanderbilt University Medical Center, Nashville, Tennessee 37232-6602
Address all correspondence and requests for reprints to: Nancy J. Brown, M.D., 560 Robinson Research Building, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6602. E-mail: nancy.j.brown{at}vanderbilt.edu.
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Abstract |
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Introduction |
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Although these effects of activation and interruption of the RAAS on the fibrinolytic system have been attributed to Ang II, increasing evidence suggests that aldosterone also regulates PAI-1 expression. First, aldosterone interacts with Ang II to increase PAI-1 expression in both vascular smooth muscle cells and endothelial cells through a mineralocorticoid receptor (MR)-dependent mechanism (9). Although the molecular basis for this interaction has yet to be defined, aldosterone enhances the vasoconstrictor (10) and fibrotic effects (11) of Ang II by increasing angiotensin II type 1 (AT1) receptor binding. In a rat model, aldosterone receptor antagonism attenuates renal PAI-1 expression after radiation injury (12). In humans, plasma PAI-1 antigen concentrations correlate with serum aldosterone concentrations in salt-depleted normal controls, hypertensive subjects, and individuals with primary hyperaldosteronism (6, 8, 9). Moreover, aldosterone receptor antagonism abolishes the relationship between aldosterone and plasma PAI-1 concentrations in hypertensive subjects (6).
Although ACE inhibition reduces PAI-1, the effect of AT1 antagonism on PAI-1 has been controversial. Data from a number of groups would suggest that short-term AT1 receptor antagonism decreases PAI-1 antigen, whereas prolonged AT1 receptor antagonism treatment does not (13, 14, 15, 16, 17, 18). Possible mechanisms for a loss of effect of AT1 receptor antagonism on circulating PAI-1 antigen concentrations over time include loss of feedback inhibition of Ang II synthesis and aldosterone escape (19, 20, 21). Moreover, given that aldosterone enhances the effect of Ang II on PAI-1 expression in vitro (9), this suggests the hypothesis that concurrent aldosterone receptor antagonism would enhance the effect of AT1 receptor antagonism on PAI-1 expression. The purpose of this study was to test that hypothesis in humans.
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Subjects and Methods |
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All subjects provided a complete medical history and underwent a physical examination before the investigation. Subjects with significant cardiovascular, renal, endocrine, or pulmonary disease or who were taking vasoactive medications were excluded. Pregnant women and women taking oral contraceptives were excluded. Written informed consent was obtained, and the study protocol was approved by the Vanderbilt University Institutional Review Board. All procedures followed were in accordance with institutional guidelines.
Protocol
Eighteen healthy subjects participated in a single blind, randomized, cross-over design study. Each subject underwent 5 study days (Fig. 1
). Twenty-four hours before each study day, subjects were asked to collect all of their urine for measurement of sodium and potassium excretion. The following morning, subjects were asked to report to the Vanderbilt General Clinical Research Center (GCRC) at 0800 h in the fasting state. An indwelling catheter was placed in an antecubital vein. BP and heart rate were measured at 0900, 1000, 1100, and 1200 h after the subject had been seated for 30 min. After each measurement of BP, blood was drawn through the indwelling catheter for measurement of PAI-1 antigen and tissue-type plasminogen activator (t-PA) antigen. Serum potassium was measured at 0900 h. Plasma renin activity (PRA), Ang II, and aldosterone were also measured at 0900 and 1000 h.
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Laboratory analysis
Blood samples were collected on ice and centrifuged immediately at 0 C for 20 min. All plasma or serum was separated and stored at -70 C until the time of assay. Blood for measurements of PAI-1 and t-PA antigen was collected in tubes containing 0.105 mmol/liter acidified sodium citrate (Biopool AB, Umea, Sweden) and antigen levels were determined using a two-site ELISA (Biopool AB). In prior studies we determined that activation and interruption of the RAAS affect PAI-1 antigen and PAI-1 activity similarly (8); therefore, PAI-1 activity was not measured. The t-PA activity was determined in the 0900 h blood sample from each study day using an immunofunctional assay (Chromolize, Biopool AB). Blood for PRA and aldosterone determinations was drawn into chilled tubes containing EDTA. PRA was measured by RIA for Ang I formation at pH 7.4 and 37 C (22). Aldosterone was measured using a commercially available RIA (Diagnostic Products, Los Angeles, CA) with an extremely low cross-reactivity to either spironolactone (0.06%) or its metabolites (below the limit of detection). Blood for Ang II determination was collected in chilled tubes containing a cocktail of protease inhibitors (23). Ang II measurements were made by RIA, as previously described (24).
Statistical analysis
Data are presented in tables as means and 95% confidence intervals unless indicated otherwise. The effect of treatment on RAAS and fibrinolytic variables was assessed using a general linear model-repeated measures ANOVA in which the within-subject variables were treatment and time and between-subject variables were quintile of body mass index and/or renin status. Post hoc pairwise comparisons were based on estimated marginal means (shown in figures and text). Adjustments for multiple comparisons were made using the method of Bonferroni. One subject suffered from symptomatic sinusitis on his fifth study day, and data from this study day were excluded from the ANOVA and replaced with the series means. A two-tailed P less than 0.05 was considered significant. All analyses were performed using SPSS for Windows (version 11.0, SPSS, Chicago).
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Results |
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Eighteen normotensive Caucasian subjects (16 males, 2 females) were studied. The mean age was 33.7 yr [95% confidence interval (CI) 29.3, 38.0)]. The mean body mass index was 26.6 (95% CI 24.7, 28.4) kg/m2. Additional baseline characteristics appear in Table 1.
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Table 1
shows the effect of treatment on mean arterial pressure (MAP). Furosemide alone did not lower MAP, compared with baseline (P = 0.146). The addition of candesartan (P < 0.001 vs. baseline and P = 0.002 vs. furosemide alone), but not spironolactone (P = 0.678 vs. baseline and P = 1.0 vs. furosemide alone), significantly lowered MAP. The blood pressure lowering effect of furosemide + combined candesartan and spironolactone was significantly greater than that of furosemide + spironolactone (P = 0.009) but not furosemide + candesartan (P = 1.0). There was no significant effect of treatment on heart rate (P = 0.090).
There was no effect of any treatment on serum potassium concentration (P = 0.689), reflecting the effectiveness of the potassium supplementation regimen outlined under Methods. Urinary potassium excretion was significantly greater during furosemide alone than during furosemide + candesartan (P = 0.023) or furosemide + spironolactone (P = 0.043) but not compared with baseline (P = 0.125) or furosemide + combination candesartan and spironolactone (P = 1.0). There was no significant effect of treatment on urinary sodium excretion (P = 0.102).
Effect of treatment on the RAAS
Figure 2 illustrates the effect of treatment on PRA, Ang II, and aldosterone concentrations. Furosemide alone significantly increased PRA [1.7 (95% CI 1.4, 2.0) vs. 1.2 (95% CI 0.9, 1.5) ng Ang I/ml·h, P = 0.028]. Addition of the AT1 receptor antagonist candesartan further increased PRA [19.3 (95% CI 8.3, 30.2) ng Ang I/ml·h, P = 0.003 vs. baseline and P = 0.003 vs. furosemide alone], whereas addition of spironolactone did not [2.3 (95% CI 1.5, 3.1) ng Ang I/ml·h, P = 0.004 vs. furosemide + candesartan]. Addition of combined treatment with candesartan and spironolactone significantly increased PRA further [25.3 (95% CI 14.1, 36.4) ng Ang I/ml·h, P < 0.001], compared with furosemide + spironolactone alone but not compared with furosemide + candesartan alone.
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Treatment with furosemide significantly increased serum aldosterone concentration [from 8.9 (95% CI 7.0, 10.9) to 14.1 (95% CI 10.9, 17.3) ng/dl, P = 0.029]. AT1 receptor blockade with candesartan significantly attenuated this effect [8.9 (95% CI 7.3, 10.6) ng/dl, P = 0.007 vs. furosemide alone], whereas MR antagonism with spironolactone [18.7 (95% CI 14.5, 22.9) ng/dl] did not. Serum aldosterone concentration was significantly higher during furosemide + combined therapy with candesartan and spironolactone [13.9 (95% CI 11.8, 15.9) ng/dl] than during furosemide + candesartan alone (P = 0.006). Aldosterone concentrations were similar during treatment with furosemide + combined candesartan and spironolactone and treatment with furosemide + spironolactone alone (P = 0.159).
Effect of treatment on fibrinolytic balance
Figure 3 shows the effect of treatment on circulating PAI-1 and t-PA antigen concentrations. Treatment with furosemide alone increased circulating PAI-1 antigen concentrations [27.8 (95% CI 20.6, 35.0) ng/ml vs. 19.3 (95% CI 13.4, 25.2) ng/ml at baseline, P = 0.002], and this effect was not blocked by administration of either the AT1 receptor antagonist candesartan [27.2 (95% CI 16.5, 37.8) ng/ml, P = 0.042 vs. baseline] or the aldosterone receptor antagonist spironolactone [27.3 (95% CI 17.9, 36.8) ng/ml, P = 0.015 vs. baseline]. In contrast, coadministration of candesartan and spironolactone [19.2 (95% CI 9.8, 28.6) ng/ml, P = 0.974 vs. baseline, P = 0.047 vs. furosemide alone] attenuated the furosemide-induced increase in PAI-1 antigen concentrations. Moreover, PAI-1 antigen concentrations were lower during furosemide + coadministration of candesartan and spironolactone than during furosemide + candesartan (P = 0.004) or furosemide + spironolactone (P = 0.030).
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The t-PA activity correlated inversely with PAI-1 antigen (R2 = 0.4321, P < 0.001). The net effect of treatment on t-PA activity is illustrated in Fig. 3C. The t-PA activity tended to be higher during furosemide + coadministration of candesartan and spironolactone than during furosemide alone (P = 0.094) and was significantly higher during furosemide + candesartan and spironolactone than during treatment with furosemide + spironolactone (P = 0.026).
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Discussion |
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Acute AT1 receptor blockade inhibits the effect of Ang II on PAI-1 expression in vascular smooth muscle cells in intact rodents and humans (18, 25, 26). Similarly, MR blockade attenuates the effect of aldosterone on PAI-1 expression in endothelial cells (9). However, with chronic AT1 receptor or aldosterone receptor antagonism in intact animals or humans, increased concentrations of Ang II or aldosterone may overcome the effects of receptor blockade on PAI-1 expression. For example, in the present study administration of candesartan significantly increased both PRA and Ang II, consistent with the loss of feedback inhibition of renin synthesis and with data from other studies of AT1 receptor antagonists (27). Likewise, as observed in recent clinical trials of spironolactone and the selective aldosterone receptor antagonist eplerenone (28), treatment with spironolactone increased Ang II and aldosterone concentrations, consistent with the loss of negative feedback of aldosterone synthesis.
Interestingly, in the case of candesartan, the marked increase in Ang II concentrations did not result in a similar increase in aldosterone, suggesting that AT1 receptor antagonism was adequate to prevent Ang II induced aldosterone synthesis. One possible explanation for this differential effect of candesartan on aldosterone and PAI-1 synthesis is that during candesartan increased Ang II stimulates PAI-1 synthesis through an AT1-independent mechanism. Ang II increases PAI-1 expression in vitro in human endothelial cells through its hexapeptide metabolite, Ang IV (5). However, the fact that acute AT1 receptor antagonism reduces PAI-1 expression in animal models (25, 26) and humans (18) as well as the observed effect of combined candesartan and spironolactone on PAI-1 in the present study argue against this explanation.
Although the lack of effect of candesartan on PAI-1 concentrations in furosemide-pretreated normotensive individuals in the present study is similar to the reported lack of effect of chronic losartan therapy on circulating PAI-1 concentrations in hydrochlorothiazide-treated hypertensive subjects (6), Fogari et al. (15) reported an increase in circulating PAI-1 during candesartan therapy in postmenopausal hypertensive women. In contrast to treatment with losartan, valsartan, or irbesartan, which did not significantly alter PAI-1 concentrations, 6- to 12-wk therapy with candesartan induced a significant 33% increase in PAI-1 antigen. The authors suggested that the difference among the AT1 receptor antagonists was related to greater activation of the RAAS (29) by the insurmountable AT1 receptor antagonist candesartan (30), compared with the surmountable antagonists such as losartan.
The lack of effect of spironolactone alone on circulating PAI-1 antigen concentrations in the present study is consistent with data from a prior study comparing the effects of the diuretic hydrochlorothiazide 25 mg/d and spironolactone 100 mg/d on fibrinolytic balance in hypertensive subjects (6). In that study, although both hydrochlorothiazide and spironolactone increased Ang II and aldosterone, only hydrochlorothiazide increased PAI-1 antigen concentrations, and spironolactone attenuated the relationship between aldosterone and PAI-1. However, interpretation of the prior study was confounded by the fact that serum potassium concentrations were higher and systolic blood pressure was lower during spironolactone treatment, compared with hydrochlorothiazide treatment. In the current study, potassium supplementation during furosemide prevented any potentially confounding differences in serum potassium concentration among treatment arms. Similarly, the coadministration of spironolactone with candesartan did not affect the hypotensive response to candesartan, excluding the possibility that hemodynamic changes could account for the effect of the two drugs on fibrinolytic balance.
On the other hand, the lack of effect of spironolactone alone on PAI-1 antigen in diuretic-pretreated subjects in the present study contrasts the data of Yalcin et al. (31), who reported that 1-wk treatment with spironolactone 50 mg/d significantly decreased PAI-1 antigen in a group of hypertensive patients. As suggested by the authors of that study, differences in volume status and the extent of activation of the RAAS may account for these disparate findings. Although we cannot exclude the possibility that higher doses of spironolactone would have suppressed PAI-1 synthesis in the current study, our previous finding that spironolactone 100 mg/d did not decrease PAI-1 antigen concentrations would not support this. Interestingly, Yalcin et al. reported that spironolactone also significantly increased t-PA antigen concentrations. In the present study, although the increase in t-PA antigen during furosemide + candesartan can be attributed to an increase in PAI-1-complexed t-PA (32), the increase in t-PA antigen during furosemide + candesartan and spironolactone, in the absence of an increase in PAI-1 antigen, suggests a genuine improvement in fibrinolytic balance, as indicated by a concurrent tendency toward increased t-PA activity.
One limitation of the present study was the use of once-daily dosing rather than twice-daily dosing of furosemide to stimulate the RAAS. Because of the short half-life of furosemide, once-daily dosing may have been inadequate to induce volume depletion and stimulate the RAAS over a 24-h period, as indicated by the lack of effect of furosemide alone on Ang II, even though PRA and aldosterone concentrations were increased. However, safety concerns regarding severe volume depletion and/or hypotension precluded the use of twice-daily dosing during concurrent candesartan and spironolactone administration. In this regard, greater activation of the RAAS during combined diuretic or combined diuretic/AT1 receptor antagonist therapy may have obscured an effect of candesartan or spironolactone alone on PAI-1. On the other hand, once-daily dosing of furosemide may more accurately reflect clinical practice.
Because the current study was designed to test the hypothesis that there is a mechanistic interaction between aldosterone receptor activation and AT1 receptor-mediated stimulation of PAI-1 expression in humans, we chose to compare the effects of an AT1 receptor antagonist and aldosterone receptor antagonist, alone and in combination, on fibrinolytic balance. However, in recent years, the Randomized Aldactone Evaluation Study (RALES) trial and other clinical trials have demonstrated an additive effect of combined ACE inhibition and aldosterone receptor antagonism on cardiovascular morbidity and mortality (33, 34). Given that ACE inhibitors decrease PAI-1 (7, 8) and that aldosterone concentrations return to baseline during chronic ACE inhibition, (20, 21), studies are needed to determine whether aldosterone receptor antagonism also enhances the effect of ACE inhibition on fibrinolytic balance. Similarly, studies are needed to assess the effect of the selective aldosterone receptor antagonist eplerenone on fibrinolytic balance. In comparison with spironolactone, eplerenone has a shorter half-life and a 10- to 20-fold lower affinity for the aldosterone receptor in vitro (35), raising the possibility that increases in circulating aldosterone concentrations could overcome any effect of this competitive aldosterone receptor antagonist on PAI-1 antigen concentrations in the setting of an activated RAAS.
In summary, the present study provides evidence for an interactive effect of Ang II and aldosterone on PAI-1 synthesis in humans. In the presence of an activated RAAS, neither AT1 receptor antagonism nor aldosterone receptor antagonism alone is sufficient to overcome the effect of increased circulating Ang II and aldosterone on PAI-1 expression. In contrast, dual AT1 and aldosterone receptor antagonism prevents the effect of activation of the RAAS on circulating PAI-1 antigen.
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Footnotes |
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Abbreviations: ACE, Angiotensin-converting enzyme; Ang II, angiotensin II; AT1, angiotensin II type 1; BP, blood pressure; MAP, mean arterial pressure; MR, mineralocorticoid receptor; PAI-1, plasminogen activator inhibitor-1; PRA, plasma renin activity; RAAS, renin-angiotensin-aldosterone system; t-PA, tissue-type plasminogen activator.
Received March 3, 2003.
Accepted May 8, 2003.
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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